Serotonin‐activated α₂‐macroglobulin inhibits neurite outgrowth and survival of embryonic sensory and cerebral cortical neurons

Abstract

Methylamine-modified alpha-2-macroglobulin (MA-alpha 2M) has been recently shown to inhibit the biological activity of beta-nerve growth factor (NGF) in promoting neurite outgrowth by embryonic dorsal root ganglia in culture (Koo PH, Liebl DJ, J Neurosci Res 31:678-692, 1992). The objectives of this study are to determine whether alpha 2M can also be modified by larger aromatic biogenic amines such as 5-hydroxytryptamine (5HT; serotonin), the nature of interaction between NGF and 5HT-modified alpha-2-M (5HT-alpha 2M), and the effect of 5HT-alpha 2M on the neurite extension and the growth of embryonic sensory and cholinergic neurons in 2 disparate animal species (chicken and rats). This study demonstrates that each mole of alpha 2M can combine with 15.2 +/- 1.8 moles of 5HT, in which up to 4.5 +/- 0.4 moles may be covalently bonded. As determined by gel filtration and polyacrylamide gel electrophoresis studies, both 5HT-alpha 2M and normal alpha 2M combine noncovalently with NGF, but 5HT-alpha 2M by comparison can combine with NGF somewhat more effectively. In contrast to normal alpha 2M, 5HT-alpha 2M at concentrations greater than about 0.17 microM exerts a dose-dependent inhibition on the NGF-stimulated neurite outgrowth by embryonic dorsal root ganglia and dissociated cells in culture, and the inhibitory effect can be overcome by higher NGF concentrations. Both 5HT-alpha 2M and MA-alpha 2M at 1.0 microM inhibit neurite extension by embryonic rat cerebral cortical cells and seriously damage these cells in culture. Such neurite-inhibitory activity, however, can only be partially blocked by extraneously added NGF alone. Normal alpha 2M (at 1.0 microM) and 5HT (at 188 microM), on the other hand, under the identical conditions produce very little or no effect on the normal cellular and axonal growth of these cells. We conclude that alpha 2M can potentially interact with nucleophilic monoamines, including neurotransmitters, to form inhibitory complexes which may inhibit/regulate NGF-promoted neurite outgrowth and neuronal survival. In addition, higher concentrations of such complexes can seriously damage certain CNS neurons which do not depend solely on NGF for survival.

Inhibition of dopamine and choline acetyltransferase concentrations in rat CNS neurons by rat ?1- and ?2-macroglobulins

“…Rat ␣ 2 M and its receptor (␣ 2 MR/LRP) have been shown to occur in various tissues including the brain (Kodelja et al, 1986;Bu et al, 1994;Higuchi et al, 1994), and rat ␣-macroglobulins, like human ␣ 2 M, can noncovalently bind to, and potentially stabilize, a variety of neurotrophins like nerve growth factor (NGF) and brain-derived growth factor (BDNF) (Koo and Stach, 1989;Liebl, 1994). Not only these properties of rat ␣ 2 M are shared by the human, but human MN-␣ 2 M are known also to exert significant effects on both PNS and CNS neurons (Koo and Liebl, 1992;Liebl and Koo, 1993a;Liebl and Koo, 1994;Hu et al, 1996b;Ç avuş et al, 1996). Since rat ␣-macroglobulins are structural and functional homologues of human ␣ 2 M, it is reasonable to assume that both rat ␣ 1 M and ␣ 2 M can also exert substantial influences upon the nervous system.…”

“…It is not surprising that both human and rat MN-␣ 2 M should have identical suppressive effects on ChAT (Fig. 2) and neurite outgrowth (Koo and Liebl, 1992;Liebl and Koo, 1993a;, since these macroglobulins are very similar antigenically, structurally as well as functionally. If ␣ 2 M is an important physiological and neurological regulator, it makes sense that it should be represented either at a high constitutive level in higher mammals such as humans (i.e., about 2 mg/ml in human serum) or as an acute-phase protein in some animals in response to stimuli.…”

“…Frontal cortices were dissected from eight to ten embryos, the tissues were dissociated by trypsinization, and the cells were combined. An aliquot containing 1 ϫ 10 7 cells was added to each 35 mm Petri plate (Falcon, Oxnard, CA) containing fresh growth media [Eagle's minimal essential medium, containing 10% fetal bovine serum, 0.1% Dglucose, 2 mM L-glutamine, and 50 µg/ml gentamicin (Sigma Chemical Co., St. Louis, MO)] (Liebl and Koo, 1993a). The cells (1 ϫ 10 7 ) were then separately incubated with PBS, BSA, rat ␣ 1 M, ␣ 2 M, 5HT-␣ 1 M, or 5HT-␣ 2 M in a final volume of 2 ml for 3 days at 37°C in a humidified 5% CO 2 atmosphere.…”

“…The physiological role of thioester bonds in ␣ 2 M homologues is unclear, but they are known to either react with entrapped proteinases or be auto-hydrolyzed subsequent to peptide bond cleavage on the ␣ 2 M site by the entrapped proteinase (Sottrup-Jensen et al, 1981;Eggertsen at al., 1991). In addition, nucleophilic amines such as methylamine and serotonin readily react with thioester bonds to form covalently linked conjugates (Swenson and Howard, 1979;Liebl and Koo, 1993a). Such proteinase and amino complexes exhibit faster electrophoretic mobilities (Barrett et al, 1979) and are capable of binding to ␣ 2 M-receptors (␣ 2 M-R/LRP), which occur in various cell types including macrophages, neurons, and astrocytes (Moestrup et al, 1992).…”

“…In addition, human MN-␣ 2 M can dose-dependently inhibit a) neurite outgrowth and survival of embryonic central nervous system (CNS) and peripheral nervous system (PNS) neurons (Koo and Liebl, 1992;Liebl and Koo, 1993a), b) choline acetyltransferase activity of basal forebrain neurons , c) dopamine (DA) release by adult caudate putamen (Hu et al, , 1996a, d) the development and maintenance of long-term potentiation in adult rat hippocampal slice (Ç avuş et al, 1996), and e) trk receptor activation and signal transduction presumably via its binding to trk . Human N-␣ 2 M, on the other hand, has little or no such effects.…”